A Functional Screen for Proteins that Alter Cell Fates

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DESCRIPTION (provided by applicant): With the complete sequence of the human and mouse genomes on the horizon, the critical next step in genomic analysis is to provide functional criteria for the newly discovered proteins. Understanding the diverse protein functions present within complex genomes must encompass many different and unique approaches. These functional approaches currently include two-hybrid screens for interacting proteins, microarray techniques for visualizing expression complexity, and other assays designed to elucidate specialized functions. We have developed one such functional-based assay that we will utilize to identify potentially hundreds of molecules that can alter specific cell fate responses. Our main focus is to understand the signals necessary for patterning and specifying diverse cellular fates during gastrulation in the mouse. Mouse gastrulation, even more so than amphibian and teleost gastrulation, is a period of vast differentiation and growth. During this stage, the mouse embryo transitions from having only two cell types, to having hundreds. Although an incredibly rich source of cell signaling, the mouse gastrula has not been used by molecular biologists to mine for molecules. This is mainly due to the size (100uM) and inaccessibility of the mouse gastrula, which therefore precludes the effective use of biochemistry, embryology and molecular assays in general. We have devised a screen that taps the identity of molecules involved in cell fate specification during mouse gastrulation. This approach delivers random combinations of cDNAs from mouse gastrula libraries into the more tractable Xenopus embryo. We then observe these embryos for changes in specific marker gene expression, indicating changes - positive or negative - in cell fate. By proceeding with a "trial run" of this screen, we have already identified 16 molecules, 8 of which have no understood function. Here, we will embark on a high throughput screen to elucidate molecules that alter neural, muscle, endothelial, blood and endodermal cell fates. At the completion of this grant we will provide the sequence, function and expression data for an estimated 200 previously unexplored molecules.